Abnormal vascular smooth muscle cell (SMC) accumulation in the intima plays a key role in the pathogenesis of atherosclerotic lesions. Locally produced insulin-like growth factors (IGFs) are important regulators of intimal SMC accumulation. IGFs stimulate SMC migration, proliferation, differentiation, and survival. These diverse actions of IGFs are mediated through the IGF-I receptor (IGF-IR), a transmembrane tyrosine kinase. How activation of the same IGF-IR by the same ligands leads to these diverse biological responses is not well understood, but is key to understanding the molecular basis of the role of the IGF signaling system in development of atherosclerotic lesions. Recently, we and others have identified several high-affinity IGF-binding proteins (IGFBPs) that are synthesized and secreted by SMCs. Our studies indicate that these IGFBPs are important determinants of specific cellular responses to IGF stimulation, and that a key player in this paradigm is IGFBP-5. IGFBP-5 binds to IGF and modulates IGF actions. IGFBP-5 also stimulates SMC migration through a ligand-independent mechanism. Our recent studies reveal that IGFBP-5 is localized in the SMC nucleus and that nuclear IGFBP-5 is likely to be derived from the secreted protein. Moreover, the conserved IGFBP-5 N-domain possesses transcriptional activation activity which is not affected by IGF binding. The overall goal of this proposal is to further elucidate the IGFBP-5 nuclear translocation pathway and to determine its role in regulating SMC migration, proliferation, differentiation, and apoptosis. The first aim will determine the membrane and cytoplasmic proteins that act as key IGFBP-5 partners and mediate IGFBP-5 internalization and nuclear translocation. The second aim will investigate the functional significance of IGFBP-5, with special focus on its nuclear targeting and ligand binding. Native and mutant IGFBP-5 will be expressed in IGFBP-5 siRNA knockdown SMCs and in IGFBP-5 null cells for in vitro studies. Transgenic mice with targeted overexpression of native or mutant IGFBP-5 in SMCs and IGFBP-5 knock-out mice will be used for in vivo studies. The third aim will determine the regulatory mechanism(s) of the transactivation activity of IGFBP-5 and to identify IGFBP-5 target genes. The proposed studies will lead us towards a better understanding of the molecular interactions between IGFs, IGF-IR and IGFBPs and provide novel information on the regulation of SMC migration, proliferation, differentiation, and apoptosis, as well as, a model of the molecular mechanisms of IGFBP-5 actions. It is our belief that elucidating the mechanisms of IGF and IGFBP-5 actions in SMCs will have important applications, including the development of future therapeutic strategies that may correct or circumvent atherosclerosis and related complications. [unreadable] [unreadable]